Toll-like receptor 4 (TLR4) and its coreceptor MD-2 mediate recognition of lipopolysaccharide (IPS), a component of all Gram negative bacteria. In the last few years it has become clear that differences in recognition of LPS structures between mouse and human TLR4/MD-2 exist. Interestingly, Yersinia pestis alters the structure of its LPS when it infects the mammalian host from its flea vector. It switches from a structure that is highly proinflammatory to one that is an antagonist on the human but not mouse receptor. To study the biological consequences of this modification we have generated a mouse model that expresses the human receptor instead of the mouse receptor. We predict this mouse will be more susceptible to Y. pestis infection. Human TLR4 polymorphisms have been shown to be associated with various diseases whereas similar coding variants in MD-2 have yet to be described. Data from the Wurfel/Martin project indicates that the D299G/T399I TLR4 variant is even less responsive to Y. pestis LPS at mammalian temperature. Therefore we are also generating a mouse model expressing this variant. These studies will determine the in vivo role of TLR4 in innate and adaptive responses to Y. pestis as well as Salmonella typhimurium, the first Gram negative bacterium shown to alter its LPS upon infecting mammalian cells. Finally, TLR4-mediated immunomodulators have been shown to protect mice from Francisella novicida or Y. pestis infection upon pretreatment with the compounds. We will therefore examine the response of the humanized mice to these TLR4 agonists. All together these experiments will determine the impact of species-specific differences in the recognition of various LPS structures by human and mouse TLR4.